A majority of antibodies raised against p-azophenylarsonate (Ars)-protein conjugates in A/J mice share a heritable cross-reactive idiotype, as they are encoded by a single combination of variable region germline gene segments termed "canonical." The dominance of the canonically-encoded structure is due to the favorable intrinsic Ars affinity of the V region germline gene combination and its ability to sustain somatic mutations conferring higher affinity leading to preferred antigen-driven selection of B cells expressing canonical V regions. The Ars systems is an important model for defining combining site structural changes occurring temporally in immune responses (affinity maturation), as functional differences among these antibodies can be related structurally by comparison to unmutated precursors. The goals of this project include: 1) To access the differentiative capacity of the germline canonical structure to generate increased affinity and to change specificity for structurally-related analogues as compared to that of a higher-affinity somatically mutated canonical antibody of known crystal structure. 2) To engineer changes in specificity and affinity ex vivo that may not be possible in vivo owing to biases inherent in the germline sequence and the somatic mutation process. A "selective" approach will be used primarily, in which libraries of mutant Fabs derived from germline (unmutated) canonical anti-Ars antibodies and high-affinity (somatically mutated) canonical anti-Ars antibodies are displayed on filamentous bacteriophage and sorted by affinity, and specificity of these mutants for Ars analogues are determined and the results interpreted in the context of the crystal structures of anti-Ars Fabs. In addition to saturation mutagenesis of different CDRs, "random" mutagenesis of entire V regions, combinatorial mutagenesis of CDRs and regions from both chains, as well as site-directed mutagenesis, will be carried out. Novel mutants will be crystallized and their structures determined. The feasibility of antibody combining site engineering to design antibodies with altered function, and in particular to increase specificity, is critical for the success of clinical immunotherapy.